We propose to determine the molecular basis for the thymidine (dThd) sensitivity of mouse EL4 thymoma cells. Cell sensitivity to dThd is thought to be a function of ribonucleotide reductase (RR) activity. We have found that the total RR activity of EL4 cells is just half the RR activity in normal cells. This is much less than the 4 to 10-fold differences found between dThd-resistant mutants and normal cells, and it is too small to account for EL4 sensitivity. However, dThd sensitivity may be a function of RR activity in nuclear polyenzyme complexes rather than total cell RR. Accordingly, we will assay RR activity in the nuclei of EL4 and normal cells to determine whether there are differences that will account for EL4 sensitivity. We will also determine whether EL4 cells, dThd-resistant EL4 mutants, and normally sensitive mouse L cells have the same levels of dThd catabolizing activity, and whether they metabolize CDP, dCDP, or dC differently in the presence of different concentrations of dThd. We will use thin layer chromatography, Dowex cation exchange chromatography, and HPLC to measure dThd-induced changes in pyrimidine metabolism in whole cells, lysolecithin-permeabilized cells, and cell lysates.